Human growth hormone (hGH), also known as somatotropin, is a single chain polypeptide hormone of 191 amino acids (molecular weight of app. 22 kD) that is synthesized in the somatotropic cells of the anterior pituitary and plays an important role in somatic growth through its effects on the metabolism of proteins, carbohydrates, and lipids. hGH is a member of a family of homologous hormones that also includes placental lactogens and prolactins [Nicoll et al., Endocr. Rev. 7(2):169-203 (1986)]. Several distinct biological activities have been ascribed to hGH, including effects on (i) linear growth (somatogenesis), (ii) lactation, (iii) activation of macrophages, and (iv) insulin-like and diabetogenic effects [Chawla et al., Annu. Rev. Med. 34:519-47 (1983); Edwards et al., Science 239(4841 Pt1):769-71 (1988); Thorner and Vance, J. Clin. Invest. 82(3):745-7 (1988)]. These biological effects derive from the interaction between hGH and specific cellular receptors, such as the growth hormone receptor [Leung et al., Nature 330 (6148):537-43 (1987)] or the prolactin receptor [Boutin et al., Cell 53(1):69-77 (1988)].
The binding of a single growth hormone (GH) molecule to a pair of GH receptors (GHR) induces receptor dimerization, promotes the rapid association of GHR with the tyrosine kinase JAK2 and activates a phosphorylation cascade involving the initial activation of the receptor-associated kinase JAK2. This results in the tyrosyl phosphorylation of the kinase itself and of the cytoplasmic domain of the receptor. The phosphorylated tyrosine residues act as docking sites for various signaling molecules that contain Src homology 2 (SH-2) or other phosphorysyl-binding domains. Among these are the STAT proteins 1, 3 and 5 (signal transducers and activators of transcription), the insulin receptor substrates (IRS) 1 and 2, which are believed to mediate some of the metabolic effects of GH and the adaptor protein Shc, leading to the activation of the Ras/MAP kinase pathway, the second messengers such as diacylglycerol, calcium, and nitic oxide. Ultimately, these pathways modulate cellular functions such as gene transcription, metabolite transport, and enzymatic activities that affect gH-dependent control of growth and metabolism. Activation by GH is very transient and several mechanisms are involved in this downregulation: internalization and degradation of the receptor and recruitment of phosphatases or of specific inhibitors of the JAK/Stat pathway, the SOCS proteins [for review, see Finidori, Vitam. Horm. 59:71-97 (2000); Carter-Su et al., Endocr. J. 43 Suppl:S65-70 (1996); Cambell, J. Pediatr. 131 (1 Pt2):S42-4 (1997)].
GH can be isolated from human pituitary glands or can be prepared recombinantly. There are two commercially available forms of the genetically engineered hormone, one of which is identical in amino acid sequence to the naturally occurring human growth hormone. The other form, isolated from a prokaryotic cell, has an additional methionine residue at the N-terminus of the protein. Recombinant forms of hGH have been available since 1993 for the long term treatment of children who have growth failure due to lack of adequate endogenous growth hormone secretion. The product is currently administered by either intramuscular or subcutaneous injection and stored in the refrigerator at 2-8° C.
GH has been either reported to have a role in, or suggested for therapy in or has shown efficacy in the treatment of (i) hypochondroplasia and idiopathic short stature [Ramaswami et al., Acta Paediatr. Suppl. 88(428):116-7 (1999); Kamp and Wit, Horm. Res. 49 Suppl. 2:67-72 (1998)]; (ii) girls with Turner syndrome [de Muinck Keizer-Schrama and Sas, Acta Paediatr. Suppl. 88)433): 126-9 (1999); Haeusler, Horm. Res. 49 Suppl. 2:62-6 (1998)]; (iii) growth delay in burned children [Low et al., Lancet 354(9192):1789 (1999); Hemdon et al., Horm. Res. 45 Suppl. 1:29-31 (1996)]; (iv) GH replacement in GH deficient adults [Bengtsson et al., J. Clin. Endocrinol. Metab. 85(3):933-42 (2000); Cook et al., Adv. Intern. med. 45:297-315 (2000); Welle, Curr. Opin. Clin. Nutr. Metab. Care 1(3):257-62 (1998); Abs et al., Clin. Endocrinol (Oxf) 50(6):703-13 (1999); Clark and Kendall, J. Clin. Pharm. Ther. 21(6):367-72 (1996)]; (v) muscle wasting under conditions, including surgical stress, renal failure, muscular dystrophy, glucocorticoid administration and HIV infection [Welle, supra; Windisch et al., Ann. Pharmacother. 32(4):437-45 (1998); Mentser et al., J. Pediatr. 131(1 Pt 2):S20-4 (1997); Hirschfeld, Horm. Res. 46(4-5):215-21 (1996)]; (vi) congestive heart failure and cardiovascular drug therapy [Cittadini et al., Miner. Electrolyte Metab. 25(102):51-5 (1999); Johnson and Gheorghiade, Am Heart J. 137(6):989-91 (1999); Sacca, Baillieres Clin. Endocrinol. Metab. 12(2):217-31; Gomberg-Maitland and Frishman, Am: Heart J. 132(6):1244-62 (1996)]; (vii) bone diseases and osteoporosis [Tanaka, Endocr. 45 Suppl:S47-52 (1998); Reginster et al., Drugs R. D. 1(3):195-201 (1999)]; (viii) puberty and reproduction [Sharara and Giudice, J. Soc, Gynecol. Investig. 4(1):2-7 (1997); Artini et al., J. Endocrinol. Invest. 19(11):763-79 (1996); Homburg, Horm. Res. 45(1-2):81-5 (1996); Homburg and Farhi, Curr. Opin. Obstet Gynecol. 7(3):220-3 (1995); Homburg and Ostergaard, Hum. Reprod. Update 1(3):264-75 (1995)]; (ix) GH therapy in elderly people [Bouillanne et al., Fundam. Clin. Pharmacol. 10(5):416-30 (1996)]; (x) wound management [Rasmussen, Dan. Med. Bull. 42(4):358-70 91995)]; (xi) breast cancer [Wennbo and Tomell, Oncogene 19(8):1072-6 (2000)]; (xii) Prader-Willi syndrome [Ritzen et al., J. Pediatr. Endocrinol. Metabol. 12 Suppl. 1:345-9 (1999); Nagai and Mori, Biomed. pharmacother. 53(10):452-4 (1999)]; (xiii) immune reconstitution [Chappel, J. Acquir. Immune Defic. Sundr. Hum. Retrovirol. 20(5):423-31 (1999)]; (xiv) obesity [Scacchi et al., Int. J. Obes. relat. Metab. disord. 23(3):260-71 (1999)]; and (xv) Russell-Silver syndrome [Stanhope et al., Horm. Res. 49 Suppl. 2:37-40 (1998)]. For further reviews on GH therapies, see Tritos and Mantzoros, Am. J. Med. 105(1):44-57 (1998); Vance, Trans. Am. Clin. Climatol. Assoc. 109:87-96 (1998); Marcus and Hoffman, Annu. Rev. Pharmacol. Toxicol. 38:45-61 (1998).
hGH is marketed under the names NUTROPIN™ or PROTROPINT™ (Genentech, Inc.), NORDOTROPIN™ (Novo Nordisk), GENOTROPIN™ (Pharmacia Upjohn), HUMATROPE™ (Eli Lilly) and SAIZEN™ or SEROSTIM™ (Serono). FDA approval is fortreatment of GH deficiency and Turner's syndrome.
To this end, variants of hGH sequences, applications and production procedures are known; see for example U.S. Pat. Nos. 4,658,021, 4,665,160, 5,068,317, 5,079,345, 5, 424,199, 5,534,617, 5,597,709, 5,612,315, 5,633,352, 5,635,604, 5,688,666 and references cited therein.
Recently, the crystal structures of wild type hGH in a 1:2 complex with its receptor was solved at 2.8 Å resolution (de Vos et al., Science 255(5042):306-12 (1992); hereby expressly incorporated by reference. The structure of this complex is deposited as 3HHR entry in the Brookhaven Protein Data Bank (PDB). The crystal structure confirmed that the complex consists of one molecule of growth hormone per two molecules of receptor. The hormone is a four-helix bundle motif characterized by the first two helices running parallel to each other but antiparallel to the last two. In addition to the structure of the wild type hGH (3HHR and 1HGU entries in the PDB), there are five crystal structures of mutant forms of hGH available in the literature and the PDB: 1HUW, 1AX1, 1A22, 1HWH, and 1HWG, hereby expressly incorporated by reference.
1HUW. PDB entry 1huw [Ultsch et al., Science 236(1): 286-299 (1994)] contains a structure (2.0 Å resolution) of a variant hGH, in which 15 mutations (F10A, M14W, H18D, H21N, K41I, Y42H, L45W, Q46W, F54P, R64K, R167N, D171S, E174S, F176Y, AND I179) were introduced with phage display mutagenesis to improve receptor binding affinity by 400-fold.
1AX1. PDB entry 1axi [Atwell et al., Science 278:1125-1128(1997)] contains a structure of a complex of a mutant of hGH (G120R, K168R, D171T, K172Y, E174A, and F176Y) with its receptor mutated at position 104: W104A. The resolution is 2.1 Å.
1A22. The PDB entry 1a22 [Clackson et al., J. Mol. Biol. 277:1111-1128 (1998)] is a structure of the 1:1 G120R growth hormone mutantreceptor complex at 2.6 Å resolution. The designed G120R mutant is an antagonist and can bind only one molecule of the GHR. 1HWH. The PDB entry 1 hwh [Sundstrom et al., J. Biol. Chem. 271(50):32197-203 (1996)] is a crystal structure of a growth hormone antagonist mutant G120R with its receptor as a 1:1 complex at 2.9 Å resolution. The 1:1 complex is remarkably similar to the native growth hormone receptor 1:2 complex. A comparison between the two structures reveals only minimal differences in the conformations of the hormone or its receptor in the two complexes, including the angle between the two immunogl;obulin-like domains of the receptor.
1 HWG. The PDB entry 1hwg (Sundstrom et al., supra) contains a crystal structure of an antagonist mutant G120R of human growth hormone in 1:2 complex with its receptor at 2.5 Å resolution. important difference between this structure and the previously published crystal structure at 2.8 Å resolution (3HHR) is revealed. Trp-104 in the receptor, a key residue in the hormone-receptor interaction has an altered conformation in the low affinity site enabling a favorable hydrogen bond to be formed with Asp-116 of the hormone.
The available crystal structure of hGH allows further protein design and the generation of more stable proteins or protein variants with an altered activity. Several groups have applied and experimentally tested systematic, quantitative methods to protein design with the goal of developing general design algorithms (Hellinga et al., J. Mol. Biol. 222: 763-785 (1991); Hurley et al., J. Mol. Biol. 224:1143-1154 (1992); Desjarlaisl et al., Protein Science 4:2006-2018 (1995); Harbury et al., Proc. Natl. Acad. Sci. U.S.A. 92:8408-8412 (1995); Klemba et al., Nat. Struc. Biol. 2:368-373 (1995); Nautiyal et al., Biochemistry 34:11645-11651 (1995); Betzo et al., Biochemistry 35:6955-6962 (1996); Dahiyat et al., Protein Science 5:895-903 (1996); Dahiyat et al., Science 278:82-87 (1997); Dahiyat et al., J. Mol. Biol. 273:789-96; Dahiyat et al., Protein Sci. 6:1333-1337 (1997); Jones, Protein Science 3:567-574 (1994); Konoi, et al., Proteins: Structure, Function and Genetics 19:244-255 (1994)). These algorithms consider the spatial positioning and steric complementarity of side chains by explicitly modeling the atoms of sequences under consideration. In particular, WO98147089, and U.S. Ser. No. 09/127,926 describe a system for protein design, both are expressly incorporated by reference.
A need still exists for proteins exhibiting both significant stability and growth hormone activity. Achievement of better stability will improve the convenience for shipment, storage and patient use of this product. Accordingly, it is an object of the invention to provide growth hormone activity (GHA) proteins with a higher thermostability than the naturally occurring hormone, nucleic acids and antibodies for the treatment of hGH related disorders.